[Ewa Krupinska]

Hello.

Have you ever had a case when you saw your expressed protein on western blot but there were no fluorescence signal?
Please help me understanding what is happening 🙂

Best.
Ewa

Ewa Krupinska
Research Engineer
Lund Protein Production Platform (LP3)
Protein Production Sweden (PPS)
Sölvegatan 35A, Lund, SE
+46 46 222 86 31
ewa.krupinska@biol.lu.se

[Rod Chalk]

I’ve seen lots of cases where there is a signal on a Western blot but no target protein detectable by mass spec. – Conclusion? I don’t trust Westerns.

[Sofia Banchenko]

Hello Ewa, could you explain, what you exactly mean? How did you see the protein on the WB? Ponceau stain? Which fluorescence is missing? Frome the HRP-tagged AntiBody (than it is probably wrong protein of same size you expecting) or your protein is tagged with the fluorecsence-protein (like GFP) and you do not see GFP? Wrong Antibody? What is the size of your protein? Best. Sofia

[Tsafi Danieli]

Hi Ewa,
Did you only express the GFP? or was the GFP at the C-term of your protein? Or is the GFP under another promoter on the construct?
The GFP levels might be too low to detect on a fluorescent microscope but sufficient to be detected on a Western, since the signal is enhanced on Westerns.

[Nick Berrow]

Hi Ewa,
Are you looking at a protein-fluorescent protein fusion or using something like EmBacY as the reporter for infection/virus generation?

If you’re looking at a fusion then probably the sensitivity of your Western will far exceed your ability to see any fluorescence, especially if you are using something like an ECL-based western protocol rather than colourimetric detection.

If you’re looking at EmBacY fluorescence as a reporter then it may be that infection levels are low and (again) the sensitivity of the Western is just too high.

I would perhaps use a Western (as well as MS) to confirm the identity of an expressed protein but am always wary of being told by researchers ‘but we can see it on a Western’ when asked to express and purify a protein. With the sensitivity of ECL-based systems ranging from pico/femtograms (some newer ones will detect attograms of protein!) they are so far removed from expression levels that make a purification viable.

We now always use a form of pull-down at small scale (pellet or media from 2-10ml of culture) with Ni/Streptactin etc. magnetic beads and SDS-PAGE with standard Coomassie staining. If nothing is detectable under these conditions then the protein is unlikely to be purifiable at larger scale.

Best
Nick

[Ewa Krupinska]

Thank you all that answered! It is actually working 🙂 (the communication within P4EU).

I should be more specific. The construct is in EmBacY so YFP signal is giving information about infection. Usually if we see our POI on WB, there is at least few cells with fluorescence. Most of the time there is even a correlation, more green signal, stronger WB band. This time, the bands are intense but there is no fluorescence signal at all ( in Hi5 cells). Not even one cell I can find on 6 well plate (2 ml of culture inside). That is something I’ve never experience before (and it has been over 10 years I’ve been working with insect cells). I am sure that virus works well as I have signal from Sf9 cells and it nicely corresponds to WB band intensity.
So I am just wondering if someone seen that before as well.

Best.
Ewa

[Kelvin Lau]

I have had this before.

In a previous life, we changed our cloning strategy to Gibson. Once in awhile we had Gibson artifacts which extended beyond our ORF and actually deleted out parts of the YFP or the promoter for it. We thus never got any fluorescence for the virus but still observed expression of protein. Until this point, we generally only Sanger Sequenced the ORF so would not have seen any changes with the rest of the backbone.

We then sequenced the plasmid from which the bacmid was from and we found the deletion. In the virus, I PCRed what shouldve been the YFP cassette, and the sequencing showed errors.

However, it can definitely be with Nick has suggested as well, that would be more plausible if you think the plasmid clone is fine.

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